Three-dimensional images reveal the impact of the endosymbiont Midichloria mitochondrii on the host mitochondria

The hard tick, Ixodes ricinus, a main Lyme disease vector, harbors an intracellular bacterial endosymbiont. Midichloria mitochondrii is maternally inherited and resides in the mitochondria of I. ricinus oocytes, but the consequences of this endosymbiosis are not well understood. Here, we provide 3D images of wild-type and aposymbiotic I. ricinus oocytes generated with focused ion beam-scanning electron microscopy. Quantitative image analyses of endosymbionts and oocyte mitochondria at different maturation stages show that the populations of both mitochondrion-associated bacteria and bacterium-hosting mitochondria increase upon vitellogenisation, and that mitochondria can host multiple bacteria in later stages. Three-dimensional reconstructions show symbiosis-dependent morphologies of mitochondria and demonstrate complete M. mitochondrii inclusion inside a mitochondrion. Cytoplasmic endosymbiont located close to mitochondria are not oriented towards the mitochondria, suggesting that bacterial recolonization is unlikely. We further demonstrate individual globular-shaped mitochondria in the wild type oocytes, while aposymbiotic oocytes only contain a mitochondrial network. In summary, our study suggests that M. mitochondrii modulates mitochondrial fragmentation in oogenesis possibly affecting organelle function and ensuring its presence over generations.

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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The 3D images and the image reconstructions generated and analysed during the current study are not publicly available due to size constrains but are available from the corresponding author on reasonable request. Exemplary electron micrographs and 3D reconstructions are represented in Figure 3. Source data for the statistical analyses are provided with this paper.

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Study description
We performed 3D image analysis of the oocytes showing the intimate interaction between the intramitochondrial endosymbiont and tick oocytes at different vitellogenisation stages. We quantitatively describe the subcellular populations of endosymbionts and mitochondria, and accurately characterize their intersections from 4 wild-type and 2 aposymbiotic tick samples. The 3D reconstructions revealed new symbiosis-dependent morphologies of mitochondria in their full architecture.

Research sample
Wild-type and aposymbiotic European hard ticks, Ixodes ricinus, are used to obtain 3D images of the oocytes. The ovaries harbour the endosymbiont Midichloria mitochondrii that is maternally transmitted. Among the egg, larva, nymph and adult stages of ticks, the adults prior to oviposition had the highest numbers of endosymbiont. Therefore, we used the semi-engorged females to harvest ovaries that carry eggs in various vitellogenic stages. The images of the oocytes in different maturation stages were acquired to monitor the interaction with the endosymbiont-mitochondrion upon egg formation. Approximately 3-5 weeks old wild-type females were collected from goats (Capra hircus) in a goat farm in Italy. The presence of the endosymbiont is confirmed by qPCR, TEM; prior to FIB-SEM imaging (n =4). The Neuchâtel line (i.e.: aposymbiotic ticks) has been reared on rabbits, and approximately 5-7 weeks old semi-engorged females are used. As the aposymbiotic ticks are very rare, no material could be used for qPCR. The absence of endosymbiont is proven by TEM; prior to FIB-SEM imaging (n = 2). Only vitellogenic and late vitellogenic stages of the posymbiotic ticks were observed to as engorgement and oviposition are drastically delayed compared to wild-type, and it was not possible to obtain a wide spectrum of maturating eggs.

Sampling strategy
No sampling procedure was used in this study, as all the samples were considered of interest due to challenges and limitations in the FIB-SEM image acquisition, and sparsity of the aposymbiotic ticks.

Data collection
Images were collected using Auriga Crossbeam Field Emission 481 Scanning Electron Microscope (Zeiss, Germany) of resin embedded samples. A 10 x 10 x 10 nm image resolution was aimed for each dataset with 2048 x 2048-pixel size in XY and milled on Z axis in 10 nm sections until the milling is interrupted. The SEM images were recorded with an aperture of 60 μm at 1.5 kV of the inlens EsB detector with the EsB grid set 488 to -300 to -500 V by Adeline Mallet and Zerrin Uzum. The images were aligned using Fiji, plugin Stacks -shuffling/Align Slices. Image reconstruction was done with original images using Amira v.2019.4 using manual segmentation tool by Zerrin Uzum. The segmentation data is transferred to Python 3.6 environment for image analysis by Dmitry Ershov ND Zerrin Uzum. prepared in October-November 2018 in Paris, France. Aposymbiotic ticks were engorged once in laboratory in Nantes, France in February 2020 and the blocks were prepared in March 2020 in Paris, France. The image processing and reconstructions were performed between July 2018-July 2020 and the statistical analysis were finalized in June-August 2020, at Institut Pasteur, France.

Data exclusions
No data are excluded.

Reproducibility
In total four independent data acquisitions and image analyses for the wild-type ticks and two for the aposymbitoic ticks were performed. All the imaging were successful. The reproducibility of the FIB-SEM images were assessed using TEM by acquisition of random section from the same sample blocks.

Randomization
The wild-type female Ixodes ricinus ticks, and the aposymbiotic Neuchatel line were randomly selected into two experimental groups prior to the beginning of the experiments. Due to the small number of biological samples and 3D data acquisitions, no randomization was performed for the imaging and image analyses.

Blinding
Due to the small number of samples, no blinding was performed during sample preparation, date collection and image reconstruction. The authors were blinded during data analysis.